Targeted protein degradation: from small molecules to complex organelles-a Keystone Symposia report.

Targeted protein degradation is critical for proper cellular function and development. Protein degradation pathways, such as the ubiquitin proteasomes system, autophagy, and endosome-lysosome pathway, must be tightly regulated to ensure proper elimination of misfolded and aggregated proteins and regulate changing protein levels during cellular differentiation, while ensuring that normal proteins remain unscathed. Protein degradation pathways have also garnered interest as a means to selectively eliminate target proteins that may be difficult to inhibit via other mechanisms. On June 7 and 8, 2021, several experts in protein degradation pathways met virtually for the Keystone eSymposium "Targeting protein degradation: from small molecules to complex organelles." The event brought together researchers working in different protein degradation pathways in an effort to begin to develop a holistic, integrated vision of protein degradation that incorporates all the major pathways to understand how changes in them can lead to disease pathology and, alternatively, how they can be leveraged for novel therapeutics.

Structural basis for membrane recruitment of ATG16L1 by WIPI2 in autophagy.

Autophagy is a cellular process that degrades cytoplasmic cargo by engulfing it in a double-membrane vesicle, known as the autophagosome, and delivering it to the lysosome. The ATG12-5-16L1 complex is responsible for conjugating members of the ubiquitin-like ATG8 protein family to phosphatidylethanolamine in the growing autophagosomal membrane, known as the phagophore. ATG12-5-16L1 is recruited to the phagophore by a subset of the phosphatidylinositol 3-phosphate-binding seven-bladedß -propeller WIPI proteins. We determined the crystal structure of WIPI2d in complex with the WIPI2 interacting region (W2IR) of ATG16L1 comprising residues 207-230 at 1.85 Å resolution. The structure shows that the ATG16L1 W2IR adopts an alpha helical conformation and binds in an electropositive and hydrophobic groove between WIPI2 ß-propeller blades 2 and 3. Mutation of residues at the interface reduces or blocks the recruitment of ATG12-5-16 L1 and the conjugation of the ATG8 protein LC3B to synthetic membranes. Interface mutants show a decrease in starvation-induced autophagy. Comparisons across the four human WIPIs suggest that WIPI1 and 2 belong to a W2IR-binding subclass responsible for localizing ATG12-5-16 L1 and driving ATG8 lipidation, whilst WIPI3 and 4 belong to a second W34IR-binding subclass responsible for localizing ATG2, and so directing lipid supply to the nascent phagophore. The structure provides a framework for understanding the regulatory node connecting two central events in autophagy initiation, the action of the autophagic PI 3-kinase complex on the one hand and ATG8 lipidation on the other.

Hydrogen Sulfide Inhibits Enzymatic Browning of Fresh-Cut Chinese Water Chestnuts.

This work investigates the role of hydrogen sulfide (H2S) in the browning and regulating the antioxidant defensive system in fresh-cut Chinese water chestnuts. The samples were fumigated with 0, 10, and 15 µl L-1 of H2S and stored at 10°C for 8 days. The results indicated that the H2S treatment significantly inhibited the browning of fresh-cut Chinese water chestnuts, reduced superoxide anion ( O 2 · - ) production rate and H2O2 content accumulation, promoted the increase of total phenol content, and enhanced activities of catalase (CAT), superoxide dismutase (SOD), ascorbate peroxidase (APX), and glutathione reductase (GR) (P < 0.05). On the other hand, phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and peroxidase (POD) activities remained at a low level in the H2S treatment (P < 0.05). This result suggested that H2S treatment might be a promising approach to inhibit browning and prolong the shelf life by enhancing oxidation resistance and inhibiting browning enzyme activity of fresh-cut Chinese water chestnuts during storage. Among them, the 15 µl L-1 H2S treatment had the best effect on fresh-cut Chinese water chestnuts.

Autophagosome biogenesis comes out of the black box.

Macroautophagic clearance of cytosolic materials entails the initiation, growth and closure of autophagosomes. Cargo triggers the assembly of a web of cargo receptors and core machinery. Autophagy-related protein 9 (ATG9) vesicles seed the growing autophagosomal membrane, which is supplied by de novo phospholipid synthesis, phospholipid transport via ATG2 proteins and lipid flipping by ATG9. Autophagosomes close via ESCRT complexes. Here, we review recent discoveries that illuminate the molecular mechanisms of autophagosome formation and discuss emerging questions in this rapidly developing field.

Reconstitution of cargo-induced LC3 lipidation in mammalian selective autophagy.

Selective autophagy of damaged mitochondria, protein aggregates, and other cargoes is essential for health. Cargo initiates phagophore biogenesis, which entails the conjugation of LC3 to phosphatidylethanolamine. Current models suggest that clustered ubiquitin chains on a cargo trigger a cascade from autophagic cargo receptors through the core complexes ULK1 and class III phosphatidylinositol 3-kinase complex I, WIPI2, and the ATG7, ATG3, and ATG12ATG5-ATG16L1 machinery of LC3 lipidation. This was tested using giant unilamellar vesicles (GUVs), GST-Ub4 as a model cargo, the cargo receptors NDP52, TAX1BP1, and OPTN, and the autophagy core complexes. All three cargo receptors potently stimulated LC3 lipidation on GUVs. NDP52- and TAX1BP1-induced LC3 lipidation required all components, but not ULK1 kinase activity. However, OPTN bypassed the ULK1 requirement. Thus, cargo-dependent stimulation of LC3 lipidation is common to multiple autophagic cargo receptors, yet the details of core complex engagement vary between the different receptors.

The autophagy adaptor NDP52 and the FIP200 coiled-coil allosterically activate ULK1 complex membrane recruitment.

The selective autophagy pathways of xenophagy and mitophagy are initiated when the adaptor NDP52 recruits the ULK1 complex to autophagic cargo. Hydrogen-deuterium exchange coupled to mass spectrometry (HDX-MS) was used to map the membrane and NDP52 binding sites of the ULK1 complex to unique regions of the coiled coil of the FIP200 subunit. Electron microscopy of the full-length ULK1 complex shows that the FIP200 coiled coil projects away from the crescent-shaped FIP200 N-terminal domain dimer. NDP52 allosterically stimulates membrane-binding by FIP200 and the ULK1 complex by promoting a more dynamic conformation of the membrane-binding portion of the FIP200 coiled coil. Giant unilamellar vesicle (GUV) reconstitution confirmed that membrane recruitment by the ULK1 complex is triggered by NDP52 engagement. These data reveal how the allosteric linkage between NDP52 and the ULK1 complex could drive the first membrane recruitment event of phagophore biogenesis in xenophagy and mitophagy.

A PI3K-WIPI2 positive feedback loop allosterically activates LC3 lipidation in autophagy.

Autophagy degrades cytoplasmic cargo by its delivery to lysosomes within double membrane autophagosomes. Synthesis of the phosphoinositide PI(3)P by the autophagic class III phosphatidylinositol-3 kinase complex I (PI3KC3-C1) and conjugation of ATG8/LC3 proteins to phagophore membranes by the ATG12-ATG5-ATG16L1 (E3) complex are two critical steps in autophagosome biogenesis, connected by WIPI2. Here, we present a complete reconstitution of these events. On giant unilamellar vesicles (GUVs), LC3 lipidation is strictly dependent on the recruitment of WIPI2 that in turn depends on PI(3)P. Ectopically targeting E3 to membranes in the absence of WIPI2 is insufficient to support LC3 lipidation, demonstrating that WIPI2 allosterically activates the E3 complex. PI3KC3-C1 and WIPI2 mutually promote the recruitment of each other in a positive feedback loop. When both PI 3-kinase and LC3 lipidation reactions were performed simultaneously, positive feedback between PI3KC3-C1 and WIPI2 led to rapid LC3 lipidation with kinetics similar to that seen in cellular autophagosome formation.

The BARA necessities of PtdIns 3-kinase activation in autophagy.

Macroautophagy/autophagy is an evolutionarily conserved degradation system with fundamental biological functions. The activation of the class III phosphatidylinositol 3-kinase (PtdIns3K) complexes and the subsequent production of phosphatidylinositol 3-phosphate (PtdIns3P) are pivotal to autophagy. Using a combination of structural biology, biochemistry, and biophysics, we revealed how the non-catalytic subunit BECN1 serves as a membrane-binding switch in the regulation of PtdIns3K complexes and autophagy.

High-Performance Nonfullerene Polymer Solar Cells Based on a Wide-Bandgap Polymer without Extra Treatment.

Nonfullerene polymer solar cells (PSCs) are developed based on a fluorinated thienyl-based wide-bandgap (WBG) polymer PBBF as the electron donor and nonfullerene small molecule IDIC as the electron acceptor. PBBF exhibits a strong absorption in the range of 300-605 nm with a wide optical bandgap of 2.05 eV, which is complementary with that of IDIC. Meanwhile, it possesses a deeper highest occupied molecular orbital energy level of  -5.52 eV and a higher hole mobility of 7.3 × 10-4  cm2 V-1  s-1 compared to the nonfluorinated polymer PBDTT. The PSCs based on PBBF:IDIC without extra treatment show a power conversion efficiency (PCE) of 8.5% with a V oc of 0.95 V, a J sc of 15.3 mA cm-2 , and an FF of 58.8%, which is much higher than that of the devices based on PBDTT:IDIC (a PCE of 5.3% with a V oc of 0.88 V, a J sc of 13.7 mA cm-2 , and an FF of 43.9%). These results indicate that PBBF is a promising WBG polymer donor material for the photovoltaic applications in nonfullerene PSCs.

Bidirectional Control of Autophagy by BECN1 BARA Domain Dynamics.

Membrane targeting of the BECN1-containing class III PI 3-kinase (PI3KC3) complexes is pivotal to the regulation of autophagy. The interaction of PI3KC3 complex II and its ubiquitously expressed inhibitor, Rubicon, was mapped to the first ß sheet of the BECN1 BARA domain and the UVRAG BARA2 domain by hydrogen-deuterium exchange and cryo-EM. These data suggest that the BARA ß sheet 1 unfolds to directly engage the membrane. This mechanism was confirmed using protein engineering, giant unilamellar vesicle assays, and molecular simulations. Using this mechanism, a BECN1 ß sheet-1 derived peptide activates both PI3KC3 complexes I and II, while HIV-1 Nef inhibits complex II. These data reveal how BECN1 switches on and off PI3KC3 binding to membranes. The observations explain how PI3KC3 inhibition by Rubicon, activation by autophagy-inducing BECN1 peptides, and inhibition by HIV-1 Nef are mediated by the switchable ability of the BECN1 BARA domain to partially unfold and insert into membranes.

AMPK-Dependent Phosphorylation of GAPDH Triggers Sirt1 Activation and Is Necessary for Autophagy upon Glucose Starvation.

Eukaryotes initiate autophagy to cope with the lack of external nutrients, which requires the activation of the nicotinamide adenine dinucleotide (NAD(+))-dependent deacetylase Sirtuin 1 (Sirt1). However, the mechanisms underlying the starvation-induced Sirt1 activation for autophagy initiation remain unclear. Here, we demonstrate that glyceraldehyde 3-phosphate dehydrogenase (GAPDH), a conventional glycolytic enzyme, is a critical mediator of AMP-activated protein kinase (AMPK)-driven Sirt1 activation. Under glucose starvation, but not amino acid starvation, cytoplasmic GAPDH is phosphorylated on Ser122 by activated AMPK. This causes GAPDH to redistribute into the nucleus. Inside the nucleus, GAPDH interacts directly with Sirt1, displacing Sirt1's repressor and causing Sirt1 to become activated. Preventing this shift of GAPDH abolishes Sirt1 activation and autophagy, while enhancing it, through overexpression of nuclear-localized GAPDH, increases Sirt1 activation and autophagy. GAPDH is thus a pivotal and central regulator of autophagy under glucose deficiency, undergoing AMPK-dependent phosphorylation and nuclear translocation to activate Sirt1 deacetylase activity.

Deacetylation of nuclear LC3 drives autophagy initiation under starvation.

Shuttling of macromolecules between different cellular compartments helps regulate the timing and extent of different cellular activities. Here, we report that LC3, a key initiator of autophagy that cycles between the nucleus and cytoplasm, becomes selectively activated in the nucleus during starvation through deacetylation by the nuclear deacetylase Sirt1. Deacetylation of LC3 at K49 and K51 by Sirt1 allows LC3 to interact with the nuclear protein DOR and return to the cytoplasm with DOR, where it is able to bind Atg7 and other autophagy factors and undergo phosphatidylethanolamine conjugation to preautophagic membranes. The association of deacetylated LC3 with autophagic factors shifts LC3's distribution from the nucleus toward the cytoplasm. Thus, an acetylation-deacetylation cycle ensures that LC3 effectively redistributes in an activated form from nucleus to cytoplasm, where it plays a central role in autophagy to enable the cell to cope with the lack of external nutrients.

Dissection of autophagy in human platelets.

Continuous turnover of intracellular components by autophagy is necessary to preserve cellular homeostasis in all tissues. Despite recent advances in identifying autophagy-related genes and understanding the functions of autophagy in developmental and pathological conditions, so far, the role of autophagy in platelet, a specific anucleate cell type, is poorly understood. In this study, we showed that human platelets express the autophagy-related proteins ATG5, ATG7, and LC3. The same as in nucleated mammalian cells, autophagy was stimulated by cell starvation or the MTOR inhibitor rapamycin in a phosphatidylinositol 3-kinase (PtdIns3K)-dependent manner. Disruption of autophagic flux led to impairment of platelet aggregation and adhesion. Furthermore, Becn1 heterozygous knockout mice displayed a prolonged bleeding time and reduced platelet aggregation. These results suggest a potential role of autophagy in the regulation of platelet function, and imply that gene transcription may not be an essential prerequisite for adaptive autophagy.

Hepatitis B virus X protein inhibits autophagic degradation by impairing lysosomal maturation.

Deficiency in autophagy, a lysosome-dependent cell degradation pathway, has been associated with a variety of diseases especially cancer. Recently, the activation of autophagy by hepatitis B virus X (HBx) protein, which is implicated in hepatitis B virus (HBV)-associated hepatocellular carcinoma (HCC), has been identified in hepatic cells. However, the underlying mechanism and the relevance of HBx-activated autophagy to the carcinogenesis caused by HBV remain elusive. Here, by transfection of HBV genomic DNA and HBx in hepatic and hepatoma cells, we showed that HBV- or HBx-induced autophagosome formation was accompanied by unchanged MTOR (mechanistic target of rapamycin) activity and decreased degradation of LC3 and SQSTM1/p62, the typical autophagic cargo proteins. Further functional and morphological analysis indicated that HBx dramatically impaired lysosomal acidification leading to a drop in lysosomal degradative capacity and the accumulation of immature lysosomes possibly through interaction with V-ATPase affecting its lysosome targeting. Moreover, clinical specimen test showed increased SQSTM1 and immature lysosomal hydrolase CTSD (cathepsin D) in human liver tissues with chronic HBV infection and HBV-associated liver cancer. These data suggest that a repressive effect of HBx on lysosomal function is responsible for the inhibition of autophagic degradation, and this may be critical to the development of HBV-associated HCC.

AP1 is essential for generation of autophagosomes from the trans-Golgi network.

Despite recent advances in understanding the functions of autophagy in developmental and pathological conditions, the underlying mechanism of where and how autophagosomal structures acquire membrane remains enigmatic. Here, we provide evidence that post-Golgi membrane traffic plays a crucial role in autophagosome formation. Increased secretion of constitutive cargo from the trans-Golgi network (TGN) to the plasma membrane induced the formation of microtubule-associated protein light chain 3 (LC3)-positive structures. At the early phase of autophagy, LC3 associated with and then budded off from a distinct TGN domain without constitutive TGN-to-plasma cargo and TGN-to-endosome proteins. The clathrin adaptor protein AP1 and clathrin localized to starvation- and rapamycin-induced autophagosomes. Dysfunction of the AP1-dependent clathrin coating at the TGN but not at the plasma membrane prevented autophagosome formation. Our results thus suggest an essential role of the TGN in autophagosome biogenesis, providing membrane to autophagosomes through an AP1-dependent pathway.